Prostate cancer is the second leading cause of cancer death among American men. The objective of this project is to develop a novel therapeutic approach to treat prostate cancer by overcoming the mechanisms of resistance. Androgen receptor (AR) signaling drives prostate cancer development and progression, and androgen deprivation therapy (ADT) is the standard of care for locally advanced and metastatic hormone nave prostate cancer (HNPC). Unfortunately, most patients develop castration resistant prostate cancer (CRPC) within 2-3 years via AR reactivation (e.g., AR overexpression, AR mutations, AR splice variants), which is also the mechanism of resistance to the two next generation anti-AR signaling inhibitors (ASI) abiraterone and enzalutamide. Further, all of these agents induce the development of AR negative neuroendocrine prostate cancer (NEPC), which constitutes approximately 25% of CRPC cases, a lethal and late stage of prostate cancer without any treatment option. Thus, there is an urgent need to develop novel targeting approaches for prostate cancer treatment. Protein arginine methyltransferase 5 (PRMT5) is an emerging oncogene that often epigenetically represses transcription of target genes such as tumor suppressors and cell cycle inhibitors in cancer cells. Preliminary findings show that PRMT5 promotes prostate cancer cell growth in vitro and in vivo via epigenetic activation of AR transcription in both HNPC and CRPC cells. Unlike ADT and ASI, PRMT5 inhibition by our novel inhibitor does not induce NEPC development in prostate cancer cells using our in vitro model system. Given that AR transcription precedes AR splicing and the translation of wild-type and mutant AR proteins, we hypothesize that targeting PRMT5 will offer an effective and unique treatment approach for prostate cancer by overcoming the mechanisms of AR reactivation without inducing NEPC development. To test this hypothesis, we will pursue the following aims. Aim 1 will determine the role of PRMT5 in regulating AR expression and cell growth in CRPC cell lines. Aim 2 will elucidate the epigenetic mechanism by which PRMT5 regulates AR transcription and cell growth as well neuroendocrine differentiation. Aim 3 will evaluate whether PRMT5 targeting demonstrates a better treatment effect for both HNPC and CRPC in animal models. Aim 4 will improve the potency and pharmacokinetic properties of PRMT5 inhibitors while preserving its specificity for prostate cancer preclinical studies. Impact: Successful completion of proposed research will gain mechanistic insight into how PRMT5 epigenetically regulates AR transcription in prostate cancer cells and will provide preclinical evidence that targeting PRMT5 is an effective approach for treatment of both HNPC and CRPC without inducing NEPC development. Further improvement and identification of potent and specific PRMT5 inhibitors will likely lead to the development of PRMT5 inhibitors for future clinical trials.